Anti-robo1 car-t cell, and preparation and application thereof

ABSTRACT

Provided is a method for modifying a chimeric antigen receptor-modified T cell (CAR-T cell). The method comprises expressing an ScFv-CD8-4-1 BB-CD3ζ molecule in a T cell. The CAR-T cell prepared using the method can specifically recognize and bind to a tumor cell with elevated expression of a ROBO1 protein, and can be used to prevent and treat a corresponding tumor-related disease

FIELD OF THE INVENTION

The present invention is related to the field of cellular drug for tumortherapy, particularly to an Anti-ROBO1 CAR-T cell, and preparation andapplication thereof.

BACKGROUND OF THE INVENTION

Human T lymphocytes recognize target cells through T cell receptors ontheir surfaces, this recognition is specific, that is, a T lymphocyterecognizes only target cells with specific antigens, and these specificantigens are presented to T lymphocytes through the action of specialmolecules after being processed in cells. These antigen-presentingmolecules are either present on the surfaces of antigen presenting-cellsor on the surfaces of target cells. There are at least two factors thatcause T lymphocytes in vivo to be unable to recognize cancer cells verywell: (1) cancer cells down-regulate the expression ofantigen-presenting molecules, and (2) the binding affinities between thepresented antigens and T cell receptors are weak. Although there are Tlymphocytes highly specific to cancer cells in cancer patients, thenumber of T lymphocytes is too small to treat cancer. Based on thissituation, scientists have proposed the concept of constructing achimeric T cell receptor (now commonly referred to as a Chimeric antigenreceptor). The Chimeric Antigen Receptor (CAR) is mainly composed of twoparts, one end of which is located outside the cell that canspecifically recognize an antigen on the surface of cancer cells, theother end of which is located in the cell that contains a signalactivation element (such as a T cell receptor, Zeta chain), which actsto transmit signals to activate T cells. Therefore, the T-lymphocytes(CAR-T cells) expressing CAR can avoid the restrictions in recognitionof the target cells by T cell, and therefore kill the target cancercells.

Currently, clinical trials of CAR-T therapy are growing rapidly, most ofwhich are evaluations of the treatment of B-cell malignancy. Mostmalignant B cell and normal B cells express the CD19 antigen, but othertypes of cells do not have CD19, so CD19 is a good therapeutic target.The compositions of CD19 CAR-T cells used in different clinical trialshave been somewhat different, and clinical designs of CD19 CAR-T cellsused in different clinical trials are also different. However, they wereall reported to have significant effects. The response rates for thetreatment of relapsed or refractory lymphocytic leukemia can reach60-90%, with some patients achieving sustained remission, the longest ofwhich was up to 2 years. Now it is not yet known that how long thesustained remission of the CD19 CAR-T treatment can achieve, but it iscertain that this kind of immunotherapy has brought effects which wereunattainable previously to some patients.

In addition to focusing on hematologic tumors, researchers also havebeen trying to extend CAR-T treatment to solid tumors. The results ofthe clinical trials have shown that GD2-specific CAR-T had certaineffects on neuroblastoma, whereas there are no therapeutic effects ofthe aFR-specific CAR-T cells on ovarian cancer, CAIX-specific CAR-Tcells on renal cell carcinoma, and PSMA-specific CAR-T cells onprostatic cancer. Carl H June et al. from the Pennsylvania Universityreported the treatment results of refractory and metastatic pancreaticductal adenocarcinoma with mesothelin-specific CAR-T cells at theAmerican Society of Clinical Oncology's annual meeting in 2015. Theresults showed that the patients had good tolerance for CAR-T cells andno cytokine syndrome occurred, CAR-T cells could be detected in theperipheral blood for a short period of time, and the conditions of 2patients were stabilized. Therefore, the use of CAR-T for the treatmentof solid tumors is still in an early stage, and there are still manyproblems to be solved.

Histopathological examination revealed that Robo1 was over expressed invarious types of cancers, such as hepatocellular carcinoma, breastcancer, colon cancer, pancreatic cancer, prostate cancer, glioma, andthe like. Studies by Ito et al. showed that Robo1 was abundantlyexpressed in hepatocellular carcinoma but only expressed in a smallamount in normal tissues, and 84.7% of liver cancer tissue samplesshowed positive expression. Therefore, Robo1 can be used as a newhepatocyte tumor-associated antigen, which is a potential therapeuticand diagnostic target. Test results of GRONE et al. showed that thecancerous tissues of 80% of colon cancer patients had high expressionlevel of Robo1 mRNA, in 45% of the patients, the expression levels were4 times over those in normal tissues, and in 15% of the patients, theexpression levels were 12 times over those in normal tissues. Therefore,Robo1 can provide a new potential target for the treatment of coloncancer. Compared with pancreatic ductal carcinoma to its surroundingbenign tissue, He et al. found that Robo1 was up-regulated in cancertissues, and this kind of up-regulation may be associated with lymphaticmetastasis of pancreatic cancer cells. Studies by Huang et al alsoshowed that Robo1 was related to the migration of colon cancer.

The extracellular domain of ROBO1 is composed of IG1-IG5 and FN3domains, with the FN3 domain being proximal to the cell membrane.Therefore, the FN3 region is a preferred choice as an antigen when ROBO1molecule is used as a target, so that when the CAR-T cells constructedby this method are in contact with tumor cells expressing ROBO1molecule, the cells would be pulled together the closest to each other,and the killing effect would be better. The specific structure is shownin FIG. 1.

SUMMARY OF THE INVENTION

The main technical problem to be solved by the present invention is toprovide an Anti-ROBO1 CAR-T cell, and preparation and applicationthereof, which is method for modifying and transforming T cells, so thatthe transformed T cells can specifically recognize and kill tumors, andthe T cells prepared by the method have more efficient tumor killingactivity.

In order to solve the above technical problem, one technical solutionadopted by the present invention is to provide a CAR-T cell targetingthe ROBO1 FN3 domain, wherein an SCFV-CD8™-4-1BB-CD3ζ fusion protein isexpressed in the T cells.

In a preferred embodiment of the present invention, the CAR-T cell ismanufactured by:

(1) synthesizing and amplifying the gene encoding theSCFV-CD8™-4-1BB-CD3ζ and cloning the gene encoding theSCFV-CD8™-4-1BB-CD3ζ fusion protein into a lentiviral expression vector;

(2) using a lentiviral envelop plasmid and the lentiviral expressionvector of step (1) to infect a 293 T cell, packaging and preparing thevirus;

-   -   (3) isolating and expanding human peripheral blood T lymphocytes        and infecting the T lymphocytes with the lentivirus of step (2)        to obtain the CAR-T cells expressing the ScFv-CD8™-4-1BB-CD3ζ        fusion protein.

In a preferred embodiment of the present invention, the SCFV sequencemolecule is expressed on a surface of the T lymphocyte, and the4-1BB-CD3ζ molecule transmits the activating signal inside the T cell.

In a preferred embodiment of the present invention, the amino acidsequence of SCFV in the SCFV-CD8™-4-1BB-CD3ζ fusion protein is SEQ IDNO:5; and the amino acid sequence of CD8¹¹ in the SCFV-CD8™-4-1BB-CD3ζfusion protein is SEQ ID NO:1.

In a preferred embodiment of the present invention, the amino acidsequence of 4-1BB in the SCFV-CD8™-4-1BB-CD3ζ fusion protein is SEQ IDNO:2; wherein the 4-1BB in the SCFV-CD8™-4-1BB-CD3ζ fusion protein canbe replaced by CD28 that has the amino acid sequence of SEQ ID NO:3.

In a preferred embodiment of the invention, the amino acid sequence ofCD3ζ in the SCFV-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:4; and the Tcell is derived from human periphery blood T lymphocytes.

In a preferred embodiment of the invention, the amino acid sequence ofthe SCFV-CD8™-4-1BB-CD3ζ fusion protein is SEQ ID NO:6.

In a preferred embodiment of the invention, the CAR-T cell is used inthe preparation anti-tumor drugs.

In a preferred embodiment of the invention, the CAR-T cell is used inpreparation of the therapeutic drugs that target tumors with highexpression of ROBO1.

The beneficial effects of the present invention are: in the Anti ROBO1CAR-T cells of the present invention, and in the preparation andapplication thereof, ROBO1 antibody is used for the construction of CARTcells, and the ROBO1 molecule is proposed as target antigen, and theAnti ROBO1 CART cells are used to kill tumor cells. In addition, theAnti ROBO1 CART cells are used as a cellular drug for the treatment oftumor diseases, which can be used for the treatment of tumors with highexpression levels of ROBO1 molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in theembodiments of the present invention, the figures used in thedescription of the embodiments will be briefly described below. It isobvious that the figures in the following description are only someembodiments of the present invention. For a person of ordinary skills inthe art, other figures can be obtained based on these figures withoutany creative work. The figures include:

FIG. 1 illustrates a schematic diagram of the structure of the ROBO1molecule of the present invention;

FIG. 2 illustrates a map of the PRRLSIN-SCFV (anti ROBO1-FN3) lentiviralplasmid vector of the present invention;

FIG. 3 illustrates a flow cytometry result of the MCF7/ROBO1 engineeringcell line with high expression of ROBO1 of the present invention.

FIG. 4 illustrates a result of the CAR-T killing experiment in vitro ofthe present invention;

FIG. 5 illustrates a result of the killing effect of CAR-T cells invitro under different effect-target ratio conditions of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The technical solutions in the embodiments of the present invention areclearly and completely described below. It is obvious that the describedembodiments are only a part of the embodiments of the present invention,not all of the embodiments. Based on the embodiments of the presentinvention, all other embodiments are obtained by a person skilled in theart without any creative work is within the scope of the protection ofthe present invention.

Example 1: Preparation of a Lentiviral Expression Vector

Synthesizing the gene encoding the SCFV (Anti ROBO1-FN3)-CD8-4-1BB-CD3ζ,wherein the gene sequence is SEQ ID NO:7, then the gene was ligated intothe PRRSLIN vector by restriction enzyme and transformation, and theupstream of the gene is EP-1α, promote. The vector was transformed intoStbl3 Escherichia coli strain and screened by ampicillin to obtainpositive clones, then the plasmids were extracted and identified byrestriction enzyme digestion, and PRRLSIN-SCFV (anti ROBO1-FN3)lentiviral transfection vector was obtain, the structure of which is asshown in FIG. 2.

Embodiment 2: Preparation of Lentivirus

(1) Twenty-four hours before transfection, seeding 293T cells into 15 cmculture dishes at a cell density of approximately 8×10⁶ cell per dish,which could ensure that the cells were at about 80% of confluence andevenly distributed in the culture dish during transfection.

(2) Prepare solution A and solution B

Solution A: 6.25 ml of 2×HEPES buffer (using 5 large dishes which arepacked together could achieve the best effects).

Solution B: adding the following plasmids respectively, and mixing:112.5 ug of pRRLSIN-EF-ROBO1 (target plasmid); 39.5 ug of pMD2.G (VSV-Genvelop); 73 ug of pCMVR8.74 (gag, pol, tat, rev); 625 μl of 2M calciumion solution. Total volume of solution A: 6.25 ml.

The solution B was mixed completely, and the solution A was addeddropwise when the solution A was gently rocked, then let the solutionsit for 5-15 minutes. The above mixed solution of A and B was gentlyrocked and added to the petri dish containing 293T cells dropwise, thenthe culture dish was gently shaken back and forth to distribute themixture of DNA and calcium ions evenly. The culture dish was placed inan incubator to incubate for 16-18 hours (do not rotate the culturedish).

Fresh medium was replaced and continued incubating, then the supernatantcontaining virus was collected after 48 hours and 72 hours,respectively. The supernatant containing virus was observed byfluorescence microscopy, more than 95% of the cells should show greenfluorescence. The supernatant was centrifuged at 500 g for 10 minutes at10° C., followed by being filtered with PES membrane (0.45 μm). BeckmanCoulter Ultra-clear SW28 centrifuge tubes were sterilized with 70%ethanol, and sterilized under UV light for 30 minutes. The filteredsupernatant containing lentivirus was transferred to a centrifuge tube.A layer of 20% sucrose was carefully spread on the bottom of thecentrifuge tube (1 ml of sucrose was added per 8 ml of supernatant). Thecentrifuge tube was equilibrated with PBS, and centrifuged thesupernatant at 25,000 rpm (82,700 g) for 2 hours at 4° C.

The centrifuge tube was carefully taken out and poured off thesupernatant, followed by being inverted to remove residual liquid. 100μl of PBS was added in the centrifuge tube and sealed, then placed at 4°C. for 2 hours, gently rocked once per 20 minutes during the time,followed by being centrifuged for 1 minute (25° C.:) at 500 g, and thevirus supernatant was collected. After being cooled on ice, the virussupernatant was stored at −80° C.

Embodiment 3

Preparation of Anti ROBO1-FN3-CART Cells:

0.5 ml of blood was taken, and tested for pathogenic microorganismsrapidly to exclude microbial infections such as HBV, HCV, HDV and HEV,HIV-1/2, Treponema pallidum and parasites; 50 ml of blood was collectedwith heparin bottle (heparin anticoagulation) under sterile conditions,and immediately (4° C., within 24 hours) sent to the cell preparationlaboratory to ensure that this process was free of pathogenic microbialcontamination. After obtaining the patient's blood, the surface of theheparin bottle was wiped with an alcohol cotton ball for disinfection inthe GMP preparation room, then the heparin bottle was placed in abiological safety cabinet. Two 50 ml centrifuge tubes were opened inadvance, and the blood was transferred into the two 50 ml centrifugetubes and tightened up. The above 50 ml centrifuge tubes filled withblood were placed in a centrifuge and centrifuged at 400 g (2000 rpm)for 10 min at room temperature, then the supernatant plasma wascollected and the precipitate layer was removed after centrifugation.The collected autologous plasma was inactivated at 56° C. for 30minutes. After being stood for 15 minutes at 4′C, the collectedautologous plasma was centrifuged at 900 g for 30 min at 4° C. to takethe supernatant for use. The enriched blood cells above were diluted to30 ml/tube with physiological saline, and two new 50 ml centrifuge tubeswere opened, then 15 ml of human lymphocyte separation liquid was addedto each centrifuge tube. The diluted blood cell solution was slowlyadded to the centrifuge tube which contains the human lymphaticseparation solution with a pipette, and tightened up. It was noted thatthe blood should be added to the upper layer of the lymphatic separationsolution, and the interface of the human lymphatic separation solutionshould not be broken. The added blood cell solution was placed in acentrifuge which was adjusted to a minimum rate of rise and fall, thenthe added blood cell solution was centrifuged at 400 g (2000 rpm) for 20min at room temperature. The middle white blood cell layer of two tubeswas collected in a 15 ml sterile centrifuge tube, and 5 ml ofphysiological saline was added, and then washed twice (Centrifuging thecollected middle white blood cell layer at 400 g for 10 minutes) toobtain peripheral blood mononuclear cells (PBMC). Complete growth mediumwas made, the concentration of V-VIVO15 added autologous AB (FBS) was5%, the concentration of IL-2 was 40 ng/ml, and the isolated PBMC wasdiluted to 2×10⁶/ml with medium, then 50 ul was taken, and the T cellspurity of PBMC was detected by flow cytometer on 0 day, Buffer1 was madethat, 1% FBS was added to PBS and the beads were rocked for 30 s ormanually shaken up and down for 5 min. CD3/CD28 beads were taken outaccording to the ratio of beads to T cells of 3-1, and the beads wereput in 1.5 ml EP tube, followed by adding 1 ml buffer1 to clean thebeads. After that, The beads were suck from the EP tube for 1 min withmagnet and washing solution was discarded, which was repeated twice,Then the beads were re-suspended to the original volume using themedium, and the cells and beads were mixed, followed by being added in asuitable culture bottle in 2×10⁶ PBMC/ML. On the second day, the densityof the cell was adjusted to 3-5×10⁶/ml, and the virus vector was addedin the proportion of virus vector:cell of 1:5, meanwhile, 4 ug/ml and 40ng/ml IL⁻² polybrene were added. After 4 hours, fresh complete mediumwas added, and the density of the cell was adjusted to 1×10⁶/ml tocontinuous culture. All the cells were centrifuged, and fresh medium wasadded to continuous culture. Half a volume change replaced per 2-3 daysto maintain the density of the cell in 0.5-1×10⁶/ml. When the number ofcells reached 10′ in the period of 10-12 days, the cells werecentrifuged at 400 g for 5 min to get inunue cells, followed by beingwashed twice with pre-cooled PBS (400 g, 5 min). The cells were count bya hemocytometer, and the cell group and the proportion of CART cellswere detected by flow cytometer. The color change, cell density, andcell morphology of the medium were observed daily and recordedaccordingly. The interleukin 2 which is required by total volume wasadded in the process of gradually expanding cultivation.

Embodiment 4

Construction and Detection of Engineering Cell Lines:

(1) Preparation of engineering cell line lentivirus with high expressionRobo1 FN3 (the specific preparing method is also the method in thesecond embodiment);

(2) Infection of MCF cells: 500,000 MCF7 cells were inoculated in 6-wellplates the day before infection. When the cells grow to 80% on the nextday, 500 μl of packaged ROBO1 virus was added in a 6-well plate,meanwhile control cell (no virus added) was set, culture medium waschanged after 12-16 hours, and then the positive cells of Robo1 weresorted by flow cytometer 3 days after infection;

(3) Detection of engineered cell lines: 20,000 cells were taken from thesorted positive cells of Robo1, followed by being centrifuged at 400 gfor 5 min, then washed twice with pre-cooled PBS, and 2.5 μl of Robo1antibody (Biolegend) was added and incubated in the dark for 20 min,after that, centrifuged and washed once with pre-cooled PBS, then thecells was re-suspended in 100 μl PBS, and the expression of Robo1 wasdetected by flow cytometer (see FIG. 3). The experimental resultsconfirmed that the engineered cell lines were successfully constructed,which can be used as a target cell for subsequent killing experiments.

Embodiment 5

Activity Assay of Anti ROBO1-FN3-CART Cells In Vitro:

LDH release assay was used to detect the killing effect of AntiROBO1-FN3-CART cells on engineered cell line MCF-1/ROBO1 and hepatomacell line SMCC7721 with high Robo1-expressing. ELISA was used to detectLDH release.

(1) Adjusting the target cells to 5×10⁴/ml with RPMI-1640 mediumcontaining 5% calf serum.

(2) Adding target cells to 96-well cell culture plates, and adding 100μl to each well. Three effector cells naturally released control wellswere only added 100 μl of culture solution without adding target cells.

(3) Adding 100 μl of effector cells to each well, and the ratio ofeffector cells to target cells was 50:1; 25:1; 10:1; 5:1; or 1:1.Natural release wells were only added 100 μl of culture medium withouteffector cells, and incubating the effector cells with the target cellsfor 6 hours, meanwhile, setting up three replicate wells for eachexperiment.

(4) Adding 10 μl Lysis Solution (10×) to the largest release well(positive control), and incubating for 45 min-60 min. Meanwhile, placingthree replicate wells each experiment.

(5) Taking out 50 ul of the test sample and the control sample in theabove 3 and 4 steps, respectively, and adding in the fresh 96-wellmicrotiter plate, then adding the assay buffer and the substance mix,followed by being protected from light for 30 minutes.

(6) Adding 50 μl stop solution.

(7) Absorbance values were measured at 490 nm or 492 nm in an hour.

(8) Killing rate=experimental group LDH (OD)/Max LDH release group (OD).

(9) Calculation formula: Killing efficiency=(experimental-effectorspontaneous−target spontaneous)/(target maximum-targetspontaneous)×100%.

The results showed that the prepared Anti ROBO1-FN3-CART cells couldsignificantly kill the target cell lines MCF-7/ROBO1 and SMCC7721 withhigh expression of ROBO1, and the different proportions of ROBO1 CAR-Tand target cells were incubated for 4 hours, followed by being detectedby ELISA experiment, which shown that the cell killing efficiency alsoincreased (see FIG. 5), and microscopic imaging showed significant deathof tumor cells (FIG. 4) with the increasing of the E:T ratio.

The above is only the embodiment of the present invention, and thus doesnot limit the scope of the patent of the present invention. Anyequivalent structure or equivalent process transformation made by usingthe content of the description of the present invention, or otherrelated technical fields were directly or indirectly applied, all thesame was included in the scope of patent protection of the presentinvention.

SEQUENCE LISTING The amino acid sequence of CD8™ SEQ ID NO: 1 is:IYIWAPLAGTCGVLLLSLVITLYC The sequence of 4-1BB SEQ ID NO: 2 is:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELThe sequence of CD28 SEQ ID NO: 3 is:RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSThe molecular sequence of CD3ζ SEQ ID NO: 4 is:RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPRThe sequence of SCFV (Anti ROBO1-FN3) SEQ ID  NO: 5 is:IQMTQTTSSLSASLGDRVTISCRASQDISNFLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDFSLTISKLEQEDIATYFCQQGNTLPLTFGAGTKLELKGGGGSGGGGSGGGGSLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKLSHGKSLEWIGDIVPNNGDTTYNQNFRGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARFSNYVYPFDYWGQGTTITVSThe sequence of SCFV (Anti ROBO1-FN3)-CD8™-4-1BB-CD3ζ fusion protein SEQ ID NO: 6 is:MALPVTALLLPLALLLHAARPIQMTQTTSSLSASLGDRVTISCRASQDISNFLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDFSLTISKLEQEDIATYFCQQGNTLPLTFGAGTKLELKGGGGSGGGGSGGGGSLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKLSHGKSLEWIGDIVPNNGDTTYNQNFRGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARFSNYVYPFDYWGQGTTITVSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRThe nucleotide sequence SCFV (Anti ROBO1-FN3)-CD8™-4-1BB-CD3ζ fusion protein SEQ ID NO: 7 is:ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCCATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTTTTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACATTCTGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTTCTCTCACCATTAGCAAACTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCACTTACGTTCGGCGCTGGGACAAAGTTGGAACTTAAAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGAGGAGGAGGATCGCTGCAACAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCTTCAGTGAAGATTTCCTGCAAGGCTTCTGGATACACATTCACTGACTACTACATGAATTGGGTGAAGCTTAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTGTTCCTAACAATGGTGATACTACTTACAACCAGAATTTCAGAGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATTCAGTAATTACGTTTACCCTTTTGACTACTGGGGCCAAGGCACCACTATCACAGTCTCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCG

1-9. (canceled) 10: A method of making an anti-ROBO1 CAR-T cell,comprising: (1) cloning a gene encoding ananti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein into a lentiviralexpression vector; (2) packaging and preparing a lentivirus byexpressing a lentiviral envelop plasmid and the lentiviral expressionvector of step (1) in a 293 T cell; (3) isolating and expanding humanperipheral blood T lymphocytes and infecting the T lymphocytes with thelentivirus of step (2) to express the ScFv-CD8-4-1 BB-CD3ζ fusionprotein in the T lymphocytes; wherein the ScFv portion of the fusionprotein is expressed on a surface of the CAR-T cell and the 4-1 BB-CD3ζportion of the fusion protein is expressed inside the CAR-T cell. 11:The anti-ROBO1 CAR-T cell of claim 10, wherein the amino acid sequenceof ScFv in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ IDNO:5; and the amino acid sequence of CD8 in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:1. 12: The anti-ROBO1 CAR-T cell ofclaim 10, wherein the amino acid sequence of 4-1BB in theanti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:2; whereinthe 4-1 BB in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein can bereplaced by CD28 that has the amino acid sequence of SEQ ID NO:3. 13:The anti-ROBO1-ScFv CAR-T cell of claim 10, wherein the amino acidsequence of CD3ζ in the anti-ROBO1-ScFv-CD8-4-1 BB-CD3ζ fusion proteinis SEQ ID NO:4. 14: The anti-ROBO1 CAR-T cell of claim 10, wherein theamino acid sequence of the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion proteinis SEQ ID NO:6. 15: The anti-ROBO1-ScFv CAR-T cell of claim 10, whereinthe T cell is derived from human periphery blood T lymphocytes. 16: Amethod of treating tumor by administering an anti-ROBO1 CAR-T cellexpressing an anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein; wherein theanti-ROBO1 portion of the fusion protein is expressed on a surface ofthe CAR-T cell and the 4-1 BB-CD3ζ portion of the fusion protein isexpressed inside the CAR-T cell. 17: The method of claim 16, wherein theamino acid sequence of ScFv in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusionprotein is SEQ ID NO:5; and the amino acid sequence of CD8 in theanti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:1. 18: Themethod of claim 16, wherein the amino acid sequence of 4-1BB in theanti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:2; whereinthe 4-1 BB in the anti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein can bereplaced by CD28 that has the amino acid sequence of SEQ ID NO:3. 19:The method of claim 16, wherein the amino acid sequence of CD34 in theanti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:4. 20: Themethod of claim 16, wherein the amino acid sequence of theanti-ROBO1-ScFv-CD8-4-1BB-CD3ζ fusion protein is SEQ ID NO:6. 21: Themethod of claim 16, wherein the tumor is characterized by highexpression level of ROBO1.